This invention relates to a process for measuring endotoxin by using a reaction of a hemacyte lysate (amoebocyte lysate) of horseshoe crab (hereinafter abbreviated as "AL") with endotoxin, in which a factor present in AL which reacts with .beta.-1,3-glucan to cause coagulation reaction (hereinafter referred to as ".beta.-1,3-glucan sensitive factor") is inactivated, and the endotoxin alone is specifically measured.
Endotoxins are lipopolysaccharides present mainly in cell wall of Gram-negative bacteria and are known as pyrogens. Therefore, the measurement of endotoxin concentration in a sample is one important measurement in the fields of medical science, pharmacy and microbiology.
At present, as a method for measuring endotoxin, the so-called Limulus test utilizing the phenomenon that an extracted AL solution (hereinafter abbreviated as "AL solution") is activated by endotoxin to form gel clot is widely employed because of its simplicity, convenience, low cost, etc.
However, it was found that AL solution reacts not only with endotoxins but also with carboxymethylated .beta.-1,3-glucan to undergo coagulation [Kakinuma et al., Biochem Biophys. Research Communication, 101 (2), 434-439 (1981)]. It was proved that this phenomenon is caused by the reaction of .beta.-1,3-glucan sensitive factor present in AL solution with .beta.-1,3-glucan or a derivative thereof (Iwanaga et al., Bacterial Endotoxin, published by Verlag Chemic, 365-382,1984).
Therefore, most of commercially available Limulus test reagents react not only with endotoxins but also with .beta.-1,3-glucan, so that it is difficult to judge which of endotoxin, .beta.-1,3-glucan and a mixture thereof is present in a sample, by the Limulus test. Thus, the specificity of such Limulus test reagents is a problem.
In order to solve this problem, there has been reported a method for preparing a reagent specific for endotoxins by removing .beta.-1,3-glucan sensitive factor from AL solution [Japanese Patent Appln. Kokai (Laid-Open) Nos. 58-13516 and 59-27828]. However, all the methods disclosed in these references require a very troublesome procedure of treating AL solution, for example, by a gel filtration method or a chromatographic method using a carrier having heparin, dextran sulfate or the like attached thereto, to separate the AL solution into a fraction of proclotting enzyme, a fraction of .beta.-1,3-glucan sensitive factor, and a fraction of a factor which reacts with endotoxin to cause coagulation (hereinafter abbreviated as "endotoxin sensitive factor"), and to remove the .beta.-1,3-glucan sensitive factor. Therefore, for preventing AL solution or the fractions obtained therefrom from being contaminated by endotoxin during the separation procedures, there are required, for example, facilities used exclusively for carrying out said procedures. Moreover, the above methods are further disadvantageous in that the individual fractions should be properly mixed again in order to obtain a reagent specific for endotoxin.
On the other hand, the above reference of Kakinuma et al. [Biochem. Biophys. Research Communication, 101 (2), 434-439 (1981)]describes the following fact. AL solution reacts not only with endotoxin but also with carboxymethylated .beta.-1,3-glucan to undergo coagulation reaction, but when carboxymethylated .beta.-1,3-glucan is added in a large amount (10 .mu.g/ml or more), it does not cause coagulation reaction, and when a large amount (10 ng/ml) of endotoxin is further added to the solution, coagulation reaction still occurred. However, the amount (10 ng/ml) of endotoxin added in said reference is as large as about 200 times the permissible concentration of endotoxin in distilled water for injection or the like [for example, the standard value (the permissible concentration in a sample) of endotoxin according to the XX edition of U.S. Pharmacopeia (USP) is or 0.05 ng/ml)]. Since such an amount is far greater than the amount which is normally measured as endotoxin using AL solution, application of the above fact for measuring endotoxin has been inconceivable.